RESUMO
Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3rd to the 5th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.
Assuntos
Códon/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Iniciação Traducional da Cadeia Peptídica , Regiões 5' não Traduzidas , Sequência de Bases , Evolução Biológica , Códon/química , Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Escherichia coli/metabolismo , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Fases de Leitura Aberta , Ribossomos/genética , Ribossomos/metabolismoRESUMO
One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.
Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Periplásmicas de Ligação/genética , Proteínas de Ligação a Fosfato/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Vibrio cholerae O1/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Códon/química , Códon/metabolismo , Biologia Computacional , Endorribonucleases/genética , Endorribonucleases/metabolismo , Sequências Repetidas Invertidas , Óperon , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vibrio cholerae O1/metabolismo , Vibrio cholerae O1/patogenicidade , VirulênciaRESUMO
Oligophenylalanines are soluble in m-cresol, but oligophenylalanyl-tRNAs are not. This differential solubility can be used to assay oligophenylalanines released from tRNA during their synthesis by wheat germ extracts. When poly U is the message, virtually no free product appears. When poly A U (A < U) is used, a considerable amount of oligophenylalanines are released. The fraction of product released is approximately constant with time, implying that a steady-state is not achieved between initiation and release. The dependence of release on various reaction variables and the effects of several inhibitors on release indicate that the reaction is probably catalyzed by peptidyl transferase, in accord with the mechanisms described for mammals and prokaryotes.